Vaia Pliaka

Abstract: Enteroviruses, the main cause of aseptic meningitis, consist of 100 serotypes, and many of them have been associated with large outbreaks. In the present study, a comparison of RFLP analysis of the 5' untranslated region (5'UTR) and sequencing of both the 5'UTR and VP1 regions was conducted for epidemiological linkage of 27 clinical enterovirus strains. The clinical enterovirus strains were clustered into five restriction profile groups. Even though the restriction profile clusters of clinical isolates were not related to those of the respective prototype strains, epidemiological relationships between the members of each cluster were observed. The restriction profile clusters in the 5'UTR corresponded to the phylogenetic clusters in the VP1 genomic region. The incongruence between the topology of Gior strain in 5'UTR and VP1 phylogenetic trees indicates a recombination event. The proposed RFLP assay in combination with VP1 sequencing can offer crucial epidemiological information about the circulating enteroviruses.

Abstract: Human noroviruses (NoVs) of the Caliciviridae family are a major cause of epidemic gastroenteritis. The NoV genus is genetically diverse and recombination of viral RNA is known to depend upon various immunological and intracellular constraints that may allow the emergence of viable recombinants. In the present study, we report the development of a broadly reactive RT-PCR assay, which allowed the characterization of strain A6 at molecular level, established its genetic relationship at the sub-genogroup level and classified A6 strain at the sub-genotype level. The detection was carried out initially by enzyme-linked immunosorbent assay (ELISA) and the subsequent detection and molecular characterization of NoV strain was achieved by reverse transcription-PCR and sequencing. Based on the sequence analysis, A6 strain was revealed to belong to the GII genogroup of NoVs. Partial ORF1 gene sequencing analysis and complete ORF2 gene sequencing revealed that ORF1 and ORF2 belonged to two distinct genotypes GII/9 and GII/6, respectively, making obvious that A6 strain is a rare intergenotypic recombinant within the genogroup GII between GII.9 and GII.6 genotypes. A6 strain represents the first human NoV from Greece, whose genome has been partially (ORF1&ORF3) and completed (ORF2) sequenced. To our knowledge the recombination event GII.9/GII.6 in RdRp and capsid gene, respectively, that was revealed in the present study is reported for the first time.

Abstract: Echovirus 6 (E6) is one of the main enteroviral serotypes that was isolated from cases of aseptic meningitis and encephalitis during the last years in Greece. Two E6 (LR51A5 and LR61G3) were isolated from the sewage treatment plant unit in Larissa, Greece, in May 2006, 1 year before their characterization from aseptic meningitis cases. The two isolates were initially found to be intra-serotypic recombinants in the genomic region VP1, a finding that initiated a full genome sequence analysis. In the present study, nucleotide, amino acid, and phylogenetic analyses for all genomic regions were conducted. For the detection of recombination events, Simplot and bootscan analyses were carried out. The continuous phylogenetic relationship in 2C-3D genomic region of strains LR51A5 and LR61G3 with E30 isolated in France in 2002-2005 indicated that the two strains were recombinants. SimPlot and Bootscan analyses confirmed that LR51A5 and LR61G3 carry an inter-serotypic recombination in the 2C genomic region. The present study provide evidence that recombination events occurred in the regions VP1 (intraserotypic) and non-capsid (interserotypic) during the evolution of LR51A5 and LR61G3, supporting the statement that the genomes of circulating enteroviruses are a mosaic of genomic regions of viral strains of the same or different serotypes. In conclusion, full genome sequence analysis of circulating enteroviral strains is a prerequisite to understand the complexity of enterovirus evolution.

Abstract: The E2 gene of human papilloma virus is expressed at the early stage of the viral life cycle, encoding the E2 transcription factor, and regulates the expression of E6 and E7 oncogenes. Disruption of E2 gene due to viral integration inhibits the transcriptional suppression of the HPV oncogenes, inducing cell proliferation. In the present study, a total of 22 HPV16-positive cytological specimens derived from high- and low-grade cervical intraepithelial lesions were investigated in order to identify sequence variations in the HPV16 E2 ORF. The E2 gene was amplified by PCR using external and internal overlapping sets of primers. Amplicons were cloned and sequenced. Disruption sites were detected in cervical samples diagnosed as high-grade cervical intraepithelial lesions. Moreover, sequence variations were identified in the E2 ORF and specific variations were associated with non-European variants such as African type I, African type II and Asian American. A total of three new sequence variations were identified at positions 2791, 2823 (transactivation domain) and 3361 (hinge region). Distinct phylogenetic branches were formed according to E2 analysis that characterized the different HPV16 variants. It was ascertained that non-European variants are circulating in the Greek population.

Abstract: The Global Polio Eradication Initiative was launched in 1988 with the aim to eliminate paralytic poliomyelitis. Two effective vaccines are available: inactivated polio vaccine (IPV) and oral polio vaccine (OPV). Since 1964, OPV has been used instead of IPV in most countries due to several economic and biological advantages. However, in rare cases, the live-attenuated Sabin strains of OPV revert to neurovirulence and cause vaccine-associated paralytic poliomyelitis in vaccinees or lead to emergence of vaccine-derived poliovirus strains. Attenuating mutations and recombination events have been associated with the reversion of vaccine strains to neurovirulence. The substitution of OPV with an improved new-generation IPV and the availability of new specific drugs against polioviruses are considered as future strategies for outbreak control and the eradication of paralytic poliomyelitis worldwide.

Abstract: The HPV16 E1(∧)E4 protein is thought to contribute to the release of newly formed viral particles from infected epithelia. In order to investigate amino acid mutations in the HPV16 E1(∧)E4 protein, the complete E4 ORF was amplified by PCR in 27 HPV16-positive cervical samples, and the amplicons were cloned. Fifteen nucleic acid variations were identified in the E4 ORF, including seven silent nucleic acid mutations. In addition, nine amino acid mutations (A7V, A7P, L16I, D45E, L59I, L59T, Q66P, S72F, H75Q) were detected in the E1(∧)E4 protein, and these were associated with the severity of cervical malignancy. A maximum-likelihood phylogenetic tree was constructed based on the E4 ORF, and nucleotide sequence analysis of the E4, E6 and E7 genes from the same samples was conducted in order to determine the phylogenetic origin of the cloned sequences from the amplified HPV16 E4. Based on the nucleotide sequence and phylogenetic analysis it was revealed that even though E4 ORF constitutes a small polymorphic portion of the viral genome (288 bp), it could provide valuable information about the origins of the HPV16 genome. In addition, molecular evolutionary analysis of the E4 coding region revealed that neutral selection is dominant in the overlapping region of the E4 and E2 ORFs.

Abstract: The molecular characterization of two enterovirus strains (LR51A5 and LR61G3) isolated from the sewage treatment plant unit in Larissa, Greece, in May and June 2006 and the investigation of their relationship with enteroviruses of the same serotype isolated in Greece in 2001 and 2007 were performed by complete VP1 sequence analysis of the isolates. The close phylogenetic relationship and the high nucleotide similarity (98%) led to the conclusion that the virus isolated from sewage in 2006 was associated with that isolated from an aseptic meningitis outbreak 1 year later. Bootscan analysis of the VP1 genomic region revealed that intraserotypic multi-recombination events might have been involved in the evolutionary past history of the LR51A5 and LR61G3 isolates.

Abstract: The live oral poliovirus vaccine (OPV) strains are genetically unstable, causing, in rare cases, vaccine-associated paralytic poliomyelitis. Reversions of the known attenuating mutations in OPV strains and intertypic recombination have been identified as the underlying causes of the increased neurovirulence of poliovirus isolates. In this study, three OPV isolates (one non-recombinant and two recombinants) were tested in order to correlate phenotypic traits such as temperature sensitivity (Rct test) and growth kinetics (one-step growth curve test) with mutations and recombination events of the viral genome. Moreover, the immunity level of the western Greek population aged 1–40 years was evaluated against OPV isolates and Sabin vaccine strains, with a microneutralization assay. Members of the 1–40-year age group (both pooled and individual sera) showed no significant differences in neutralization test (NT) titres against OPV isolates in comparison with the Sabin vaccine strains. However, all three OPV isolates showed reverted phenotypic traits in Rct or one-step growth curve assays. The results of our study revealed a significant decrease in immunity level from the 1–10-year age group to the 21–30-year age group (pooled sera) for both poliovirus types 1 and 3. For both poliovirus types, the highest NT titres were observed in the 1–10-year age group, and the lowest NT titre was observed in the 21–30-year age group, towards poliovirus type 3. Our study underlines the need for immunological studies in all age groups, in order to allow reconsideration of the current vaccination policies and to avoid epidemics caused by the circulation of highly evolved OPV derivatives.

Abstract: In this study, the immunity level of the southern Greek population in the 1–10-year, 11–20-year, 21–30-year and 31–40-year age groups with regard to Sabin vaccine strains and a collection of 11 recombinant and three non-recombinant poliovirus vaccine strains was determined. The results showed the lowest neutralization titre in the 21–30-year-age group against poliovirus type 3. Moreover, the capsid coding region of OPV (oral poliovirus vaccine) derivatives was sequenced in order to identify mutations that might lead to antigenic changes. In Sabin-1 derivatives a tendency of accumulation of mutations was observed in or near antigenic sites while in Sabin-2 and Sabin-3 derivatives in sites known to be involved in restoring neurovirulence or eliminating their temperature-sensitive phenotype. It was concluded that the combination of mutations in the capsid coding region and not the number of specific mutations in antigenic sites determines the antigenic properties of OPV derivatives and their reactivity with human sera.

Abstract: The molecular characterization of two enterovirus strains (LR51A5 and LR61G3) isolated from the sewage treatment plant unit in Larissa, Greece, in May and June 2006 and the investigation of their relationship with enteroviruses of the same serotype isolated in Greece in 2001 and 2007 were performed by complete VP1 sequence analysis of the isolates. The close phylogenetic relationship and the high nucleotide similarity (98%) led to the conclusion that the virus isolated from sewage in 2006 was associated with that isolated from an aseptic meningitis outbreak 1 year later. Bootscan analysis of the VP1 genomic region revealed that intraserotypic multi-recombination events might have been involved in the evolutionary past history of the LR51A5 and LR61G3 isolates.

Abstract: Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and in rare cases may cause vaccine-associated paralytic poliomyelitis (VAPP). Mutations at specific sites of the genome and recombination between Sabin strains may result in the loss of the attenuated phenotype of OPV (Oral Poliovirus Vaccine) strains and the acquisition of traits characteristic of wild polioviruses, such as increased neurovirulence and loss of temperature sensitivity. In this study, we determined the phenotypic traits such as temperature sensitivity and growth kinetics of eight OPV isolates (six bi-recombinant and two non-recombinant). The growth phenotype of each isolate as well as of Sabin vaccine strains in Hep2 cell line at two different temperatures (37 and 40C) was evaluated using two different assays, RCT test (Reproductive Capacity at different Temperatures) and one-step growth curve analysis. Moreover, the nucleotide and amino acid positions in the genomes of the isolates that have been identified as being involved in the attenuated and thermo sensitive phenotype of Sabin vaccine strains were investigated. Mutations that result in loss of the attenuated and thermo sensitive phenotype of Sabin vaccine strains were identified in the genomes of all isolates. Both mutations and recombination events correlated well with the reverted phenotypic traits of OPV-derivatives. In the post-eradication era of wild polioviruses, the identification and the characterization (genomic and phenotypic) of vaccine-derived polioviruses become increasingly important in order to prevent cases or even outbreaks of paralytic poliomyelitis caused by neurovirulent strains.

Abstract: In the post-eradication era of wild polioviruses, the only remaining sources of poliovirus infection worldwide would be the vaccine-derived polioviruses (VDPVs). As the preponderance of countries certified to be polio-free has switched from OPV (oral poliovirus vaccine) to IPV (inactivated poliovirus vaccine), importation of recombinant evolved derivatives of vaccinal strains would have serious implication for public health. To test the robustness of the proposed RT-PCR screening analysis, eleven recombinant vaccine-derived polioviruses that were characterized previously by sequencing by our group, in addition to three recently identified recombinant environmental isolates were assayed. Although the most definitive characterization of VDPVs is by genomic sequencing, in this study we describe a new, inexpensive and broadly applicable RT-PCR assay for the identification of the predominant recombination types S3/Sx in 2C and S2/Sx in 3D genomic regions respectively of VDPVs, that can be readily implemented in laboratories lacking sequencing facilities as a first approach for the early detection of vaccine-derived poliovirus (VDPVs).

Abstract: In this study, the serological status of the southern Greek population in the 1–10-year, 11–20-year, 21–30-year and 31–40-year age groups with regard to Sabin vaccine strains and a collection of 15 recombinant and four non-recombinant poliovirus vaccine strains was determined. For all three poliovirus types, the highest neutralization test (NT) titres were observed in the 1–10- year age group, indicating a good response to vaccination. In general, the serological status of the population of southern Greece with regard to poliovirus is better for types 1 and 2 than for type 3. The presence of the lowest NT titre in the 21– 30-year age group against poliovirus type 3 suggests the need for a booster dose of monovalent Sabin3 vaccine to ensure personal and herd immunity.

Abstract: Two enteroviruses from river water and four from sewage treatment plant were isolated in Larissa, Greece, that all shared the same sequence. A full genome analysis was conducted in an attempt to reveal the evolutionary pathways of one of the isolated strains (LR11F7). VP1 nucleotide and phylogenetic analysis revealed that the isolated strain had 78% homology with the echovirus 7 prototype strain Wallace. Full genome analysis revealed that LR11F7 P1 region is related to echoviruses 7 and that P2 and P3 regions are originating from contemporary enteroviruses isolated in South Asia. Two recombination events were shown to be involved into the evolutionary history of LR11F7, the one event concerning 3A, 3B, and 2C, and the other concerning 3D genomic region, both with new types of HEV-B. The contribution of recombination to enterovirus evolution is substantial, giving rise to new genetic lineages with unknown properties.

Abstract: Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

Abstract: An echovirus 3 (Echo3) strain (strain LR31G7) was isolated from a sewage treatment plant in Greece in 2005. Full-genome molecular, phylogenetic, and SimPlot analyses were conducted in order to reveal the evolutionary pathways of the isolate. Nucleotide and phylogenetic analyses of part of the VP1 genomic region revealed that the isolated strain correlates with Echo3 strains isolated during the same year in France and Japan, implying that the same virus circulated in Europe and Asia. LR31G7 was found to be a recombinant that shares the 3 part of its genome with an Echo25 strain isolated from asymptomatic infants in Norway in 2003. Nucleotide and SimPlot analyses of the VP1-2A junction, where the recombination was located, revealed the exact recombination breakpoint (nucleotides 3357 to 3364). Moreover, there is evidence that recombination events had occurred in 3B-3D region in the evolutionary history of the isolate. Our study indicates that recombination events play major roles in enterovirus evolution and that the circulation of multirecombinant strains with unknown properties could be potentially dangerous for public health.

Abstract: In order to test the hypothesis that RNA structural elements promote the distribution of certain types of recombination junctions in each one of the 2C and 3D poliovirus genomic regions (Sabin 3/Sabin 2 or Sabin 1 in 2C and Sabin 2/Sabin 1 or Sabin 3 in 3D), we searched in 2C and 3D regions of reference Sabin strains for high probability RNA structural elements that could promote recombination. Recombination junctions that were identified in clinical strains of this study, as well as in clinical strains of previous studies, were superimposed on RNA secondary structure models of 2C and 3D genomic regions. Furthermore, we created an in vitro model, based on double infection of cell-culture with two poliovirus strains, for the production and identification of recombinant Sabin strains in 2C and 3D regions. Our intention was to compare the results that refer to the correlation of recombination junctions and RNA secondary structures in 2C and 3D regions of clinical strains, with the respective results of the in vitro model. Most of the recombination junctions of the clinical strains were correlated with RNA secondary structure elements, which were identical between recombining Sabin strains, and also presented high predictive value. In consensus were, the respective results originated from the in vitro model. We propose that the distribution of specific types of recombination junctions in certain regions of Sabin strains is not fortuitous and is correlated with RNA secondary structure elements identical to both recombination partners. Furthermore, results of this study highlight an important role for the stem region of the RNA structure elements in promoting recombination.

Abstract: The genetic properties of strain K/2002, isolated from fecal samples of a 7-month-old child who had received his first oral poliovirus vaccine (OPV) dose at the age of 3 months, are described. Preliminary sequencing characterization of isolate K/2002 revealed an S3/S2 recombination event at the 3' end of the VP1 coding region. A recombination event resulted in the introduction of six Sabin 2 amino acid residues in a Sabin 3 genomic background. Furthermore, mutations associated with loss of the attenuated phenotype of Sabin 3 strains have been identified in the genome of isolate K/2002. The data presented here emphasize the need for careful planning of vaccination strategies, which involve stopping OPV administration in regions that are certified to be polio-free.

Abstract: Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several enteroviruses serotypes are not easily isolated in cell cultures system used and routinely more than one passage in cell culture is performed. A total of 54 archived faecal samples were examined. The heterogeneous nature of faecal samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. PCR inhibitors are frequently encountered in stool specimens. From the three methods initially compared for extraction of viral RNA, QIAamp Viral RNA Mini Kit was retained as it yielded the highest amount of viral RNA without the interference of RTPCR inhibitors. Evaluation of 54 archived stool specimens by RT-PCR and cell culture resulted in a higher frequency of detection by RT-PCR. With the use of RT-PCR we were able to detect two additional samples otherwise considered negative for enterovirus isolation if only the cell culture standard methodology was employed. RNA extraction with QIAamp Viral RNA Mini Kit coupled with RT-PCR in the 50NCR (subgrouping into distinct genetic clusters of all enteroviruses) and VP1 (reliable serotyping by sequencing) is a rapid and sensitive technique of direct poliovirus/non-polio enteroviruses recovery and molecular characterization from human faecal specimens without further passage in cell culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Abstract: Polioviruses are members of the enterovirus genus, belonging to the Picornaviridae family. They are the causative agents of poliomyelitis, a paralytic and sometimes fatal disease in humans. The number of poliomyelitis cases caused by wild poliovirus infections has been dramatically reduced by the extensive use of two available vaccines: the inactivated poliovirus vaccine (IPV) and the oral poliovirus vaccine (OPV). Despite the importance of OPV in the reduction of poliomyelitis cases, one of the disadvantages associated with this vaccine is the rare occurrence of vaccine-associated paralytic poliomyelitis (VAPP) in vaccinees or their healthy contacts through the accumulation of mutations and/or recombination in Sabin strains genome. Thirteen clinical isolates originating from healthy vaccinees and VAPP cases were investigated in order to identify genomic modifications in 5' non-coding region (5'-NCR) and VP1 genomic regions. The analysis of samples was conducted by RT-PCR, RFLP, sequencing and bioinformatics analysis. All clinical isolates were characterized as OPV-like viruses. Our results showed that analysis of 5'-NCR and VP1 regions of Poliovirus Sabin strains is important in order to identify mutations that increase the neurovirulence conducting to the eventuality of emergence of VAPP cases.

Abstract: Retrospective molecular and phenotypic characterization of a vaccine-derived poliovirus (VDPV) type 1 isolate (7/b/97) isolated from sewage in Athens, Greece, in 1997 is reported. VP1 sequencing of this isolate revealed 1.87% divergence from the VP1 region of reference strain Sabin 1, while further genomic characterization of isolate 7/b/97 revealed a recombination event in the nonstructural part of the genome between a vaccine strain and a nonvaccine strain probably belonging to Enterovirus species C. Amino acid substitutions commonly found in previous studies were identified in the capsid coding region of the isolate, while most of the attenuation and temperature sensitivity determinants were reverted. The ultimate source of isolate 7/b/97 is unknown. The recovery of such a highly divergent derivative of a vaccine strain emphasizes the need for urgent implementation of environmental surveillance as a supportive procedure in the polio surveillance system even in countries with high rates of OPV coverage in order to prevent cases or even outbreaks of poliomyelitis that otherwise would be inevitable.